PROJECT SUMMARY MicroRNAs (miRNAs) are noncoding RNAs that regulate gene expression in eukaryotic species. Their precursors have a stem-loop structure, and Dicer measures the distance from the 3' 2- nucleotide (nt) overhang when cropping the loop region. The resultant 19~23-nt miRNA duplexes are loaded into Argonaute proteins (AGOs). Therefore, miRNAs are defined by the size of 19~23 nucleotide (nt) length. Most of the early studies about miRNAs using next-generation RNA sequencing (RNAseq) excluded ~18 nt short RNAs, and thus little is known about such tiny RNAs (tyRNAs). However, recent studies reported that many tyRNAs actually bind to AGOs, although their physiological rule remains unknown. The long-term goal of this project is to understand the physiological role of tyRNAs comprehensively and to determine their biogenesis pathways. The short-term objective is to focus on a specific type of tyRNAs capable of converting AGO3 to a slicer like AGO2. Such tyRNAs were discovered by our preliminary studies and named `cleavage-inducing tyRNAs (cityRNAs).' In addition, we also identified a nuclease that trims AGO3-bound guide RNA to a 14 nt cityRNA, thereby activating AGO3 for RNA cleavage. In this study, we hypothesize that several nucleases shorten AGO-bound miRNAs to tyRNAs, some of which work as cityRNAs to catalytically activate AGO3. To validate this hypothesis, we will pursue the following specific aims. In Aim 1, cleavage assays using different guide RNAs and AGO3 mutants will be used to determine the requirements of cityRNA and AGO3 for target cleavage. We will also determine the crystal structures of AGO3 in complex with cityRNAs, which will provide the structural basis for the recognition of cityRNAs by AGO3. In Aim 2, cleavage assays using different targets will be used to determine the requirements of target RNAs for cleavage by cityRNA-loaded AGO3. We will solve the crystal structures of AGO3 in complex with cityRNAs and their target RNA, which will elucidate the mechanism of the target recognition. In Aim 3, RNAseq and transcriptome analyses will determine the endogenous cityRNAs and targets cleaved by AGO3 in the innate immune response. Altogether, outcomes from this study will reveal the molecular mechanism of cityRNA-directed RNA cleavage and the correlation between cityRNAs and innate immune response.